HIV-1 protease monomer consists of a 99 amino acid polypeptide with two reactive cysteine residues (Cys 67 and Cys 95) which may be involved in a dimerization process to activate the protease catalytic cavity. Protoniation of Asp 25 and Asp 25' residues mediates the cleavage of the gag and gag-pol polyproteins in the process of virion maturation, which is essential for virus infectivity. Several aspartyl protease inhibitors have been developed as critical anti-retroviral agents used in the cocktail treatment of AIDS. However, their use eventually leads to development of drug-resistant HIV strains. The development of HIV resistance to protease inhibitors may be due in part to the mutation of the protease. Thus there is an urgent need to find novel protease inhibitors that are active against mutated strains of HIV with their resistance to available drugs. We have recently reported that nanomolar HIV-1 protease causes significant neurotoxicity that can be prevented not only by aspartyl protease inhibitors (i.e., KNI-272, indinavir) but also S-nitrosoglutathione (GSNO) at micromolar concentrations. Preliminary data indicate that GSNO concentration-dependently inhibited the proteolytic activity of wild type HIV-1 protease, and it was 2 to 3 orders of magnitude more potent than other nitric oxide (NO) donors (i.e., SNAP) and thiol agents (i.e., GSSG). Based on these and other observations I propose that micromolar GSNO may interact with cysteine residues at the dimeric interface through the modification of sulfhydryl group, altering the 3-D configuration of HIV-1 protease homodimer thus inhibiting its proteolytic activity. GSNO, but not NO-exhausted oxidized GSNO, inhibited wild type HIV-1 protease activity in a concentration-dependent manner. This anti-protease effect of GSNO is at least two to three orders of magnitude more potent than other NO donors and thiol reagents. Experiments using protease cysteine mutants C67A and C95A demonstrated that GSNO inhibits protease activity by acting through the modification of Cys 95 at the dimeric interface. This unique anti-protease mechanism of GSNO is completely different from current aspartyl protease inhibitors since the anti-protease effect of GSNO was prevented and reversed by thiol reducing agents such as dithiothreitol and tris(2-carboxyethyl)phosphine. These new observations suggest that GSNO-derived protease inhibitors may have utility as a new class of HIV-1 protease inhibitor.